This invention pertains to preparation and use of very high surface area porous substrates that can be used to synthesize high density arrays of polymers.
Porous silica glass has been known for quite some time. U.S. Pat. No. 4,220,461 provides a historical perspective and discussion on the development of silica-rich phase-separable porous glass. Various methods for the manufacture of phase-separable porous glass are reviewed in U.S. Pat. No. 4,528,010. Both of these references are incorporated by reference in their entireties for all purposes.
The present invention relates to a porous substrate and methods for making and using the porous substrate. The porous substrate provides an increased surface area for polymers to attach to the substrate. Such porous substrates are often used to make an array of polymers, such as for genetic diagnostic purposes. The polymers may be placed or fabricated on the porous substrate by various methods.
The polymers can include those of biological interest such as nucleic acids, polynucleotides, proteins, polypeptides, polysaccharides, oligosaccharides, mixtures of these or other polymers on an array and combinations of the above polymer units in individual polymers. The porous substrates thus are useful in, for example, glass technology, polymer chemistry, molecular biology, medicine, and medical diagnostics.
The porous substrate generally has at least two regions, a support region and a porous region. The support region, which can serve as an underlayer region, basically provides mechanical support for ease of handling of a porous region. The porous region may be for example a layer (film). The support region can be selected or processed to provide additional features in the finished porous substrate. One advantage of using a porous region with higher surface area to make an array is that the array can be functionalized with a much higher density of polymers for a given two dimensional area without changing the spacing between polymers on the surface of the porous substrate.
One embodiment of this invention provides a primarily inorganic porous substrate including a support region, and a porous region in contact with the support region. The porous region for example includes pores with a pore size of 1-500 nm, or 2-500 nm, the porous region having a porosity of, e.g., 10-90%, 20-80%, or 70-90%, and a porous surface thickness of 0.01-20 xcexcm, wherein the porous region has a surface capable of forming arrays of polymers thereon. The porosity is generally, for example, xe2x80x9copenxe2x80x9d, that is, some pores are connected to others to allow the infusion of polymers or other fluids. Not all the pores need to connect to another, that is, some of the pores may be closed. What is meant by xe2x80x9cprimarily inorganicxe2x80x9d is that a small amount of organic material may remain in the porous region of the substrate, or may be intentionally applied onto the surface(s) of the porous region.
In one embodiment, a porous substrate is provided comprising:
a support region; and
a porous region on the support region, the porous region being primarily inorganic and having a surface capable of forming a polymer array thereon, the porous region comprising pores of a pore size of about 2 nm-500 nm or 1000 Angstroms to 500 nm, a porosity of about 10-90%, and a thickness of about 0.01 xcexcm to about 70 xcexcm.
The porous region can be formed by an additive method, which can include the application of colloidal silica on the support region. The additive method also may include the application of alkoxysilane on the support region. The porous region may comprise silica. The porous region may further comprise organic polymer of less than or equal to about 10% mole fraction. The porous region may comprise a plurality of pores, each of the plurality of pores having a size of from about 2 to about 100 nm. The porous region may comprise a plurality of pores, each of the plurality of pores having a size of from about 2 to about 50 nm. The porous region has, for example, a porosity of from about 20-80%, or 50-70%. The porous region for example comprises a plurality of particles, each of the plurality of particles having a size from about 5-500 nm, 5-200 nm, or 70-100 nm. The porous region has, for example, a thickness from about 0.1-1 microns, or about 0.1 xcexcm to about 0.5 xcexcm, or about 1 xcexcm to about 20 xcexcm.
An organic polymer may coat silica particles of the porous region. The porous region may be silylated with a silyating agent, such as N,N-bis(hydroxyethylaminopropyl)triethoxysilane and glycidoxypropyl trimethoxy silane. The porous region may be formed by codepositing an organic template material with silica, followed by removing the organic template material. The organic template material for example comprises particles of about 10-100 nm and the silica comprises particles of about 7-100 nm. The organic template particle size can be about equal to a silica particle size. The silica particle size is for example less than or equal to about ⅔ an organic template particle size. The silica particle size is in one embodiment, less than about 10% of an organic template particle size. The organic template material can be deposited in a volume ratio to the silica of about 10:1 to 1:10, e.g., 2:1. The organic template material is in one embodiment removed using a baking process at a temperature of above about 150xc2x0 C. The silica may be densified using an annealing process. The porous region has in one embodiment an effective surface area about 15-40 times a flat substrate with an equivalent two dimensional structure. In one embodiment, the porous region is formed by a subtractive method. The organic template polymer may be a latex polymer. The porous substrate may comprise phase-separable glass, a surface portion of the phase-separable glass being treated to form the porous layer. The phase-separable glass may comprise for example a sodium borosilicate glass. The sodium borosilicate glass may be been annealed and leached to provide the porous layer having a thickness of about 70 microns and comprised of a plurality of pores, at least some of the plurality of pores having a pore size greater than about 1000 xc3x85. The porous region has, e.g., an effective surface area about 50-400 times a flat substrate with an equivalent two dimensional structure.
The porous substrate may further comprise a high density array of polymers, such as nucleic acids immobilized on the surface.
In another embodiment, a porous substrate is provided comprising:
a support region; and
a porous region on the support region, said porous region being about 0.1-0.5 microns thick,
wherein the porous layer comprises an unsintered matrix formed from at least colloidal silica having a particle size of about 70-100 microns, the unsintered matrix defining at least a plurality of open pores having a pore size of about 10-20 nm, and
wherein the porous layer has a porosity of of about 10-90%.
In one embodiment, a method of forming a porous substrate is provided, the method comprising:
providing a substrate material comprising a surface;
dipping the substrate material in a solution including colloidal silica and a carrier, the colloidal silica having a particle size of about 12-100 nm; and
withdrawing the substrate material to provide an unsintered porous layer having a thickness of about 0.1-1 microns and a porosity of of about 10-90% on the substrate material.
Also provided is a method of forming a porous substrate, the method comprising:
providing a substrate material comprising a surface;
applying a solution including colloidal silica and a carrier to the surface of the substrate material, the colloidal silica having a particle size of about 12-100 nm;
spinning the substrate material and the applied solution to achieve a spun layer on the substrate material; and
removing the carrier from the spun layer to provide an unsintered porous layer having a thickness of about 0.1-1 microns and a porosity of about 10-90% on the substrate material.
Another embodiment is a method of forming a porous substrate comprising different monomer sequences, the method comprising:
immobilizing different monomer sequences on a porous substrate.
In another embodiment, there is provided a method of synthesizing polymers on a porous substrate, the method comprising:
a) generating a pattern of light and dark areas by selectively irradiating at least a first area of a surface of a porous substrate, said surface comprising immobilized monomers on said surface, said monomers coupled to a photoremovable protective group, without irradiating at least a second area of said surface, to remove said protective group from said monomers in said first area;
b) simultaneously contacting said first area and said second area of said surface with a first monomer to couple said first monomer to said immobilized monomers in said first area, and not in said second area, said first monomer having said photoremovable protective group;
c) generating another pattern of light and dark areas by selectively irradiating with light at least a part of said first area of said surface and at least a part of said second area to remove said protective group in said at least a part of said first area and said at least a part of said second area;
d) simultaneously contacting said first area and said second area of said surface with a second monomer to couple said second monomer to said immobilized monomers in at least a part of said first area and at least a part of said second area; and
e) performing additional irradiating and monomer contacting and coupling steps so that a matrix array of different polymers is formed on said surface, whereby said different polymers have sequences and locations on said surface defined by the patterns of light and dark areas formed during the irradiating steps and the monomers coupled in said contacting steps.
The monomers are for example, nucleotides, amino acids, or monosaccharides. The substrate may have linker molecules on its surface.
There also is provided a method of forming polymers having different monomer sequences on a porous substrate, the method comprising:
providing a porous substrate comprising a linker molecule layer thereon, said linker molecule layer comprising a linker molecule and a protective group;
applying a barrier layer overlying said linker molecule layer, said applying step forming selected exposed regions of said linker molecule layer;
exposing said selected exposed regions of said linker molecule layer to a deprotecting agent to remove the protective group; and
coupling selected monomers to form selected polymers on the substrate.
The deprotection agent may be, for example, in the vapor phase or liquid phase, and may be, for example an acid, such as trichloroacetic acid, dichloroacetic acid, or HCl. The monomers are for example nucleotides, amino acids, or monosaccharides.
In another embodiment, there is provided a method for detecting a nucleic acid sequence, the method comprising:
(a) providing an array of nucleic acids bound to the porous substrate;
(b) contacting the array of nucleic acids with at least one labeled nucleic acid comprising a sequence substantially complementary to a nucleic acid of said array, and
(c) detecting hybridization at least the labeled complementary nucleic acid to nucleic acids of said array.
In one embodiment, the porous substrates comprising arrays may be used to screen for a previously identified polymorphic variant in a target nucleic acid sequence, or for a target such as a human immunodeficiency virus sequence. Nucleic acids such as a p53 gene, an HIV RT gene, a CFTR gene, or a cytochrome p450 gene can be screened for. The array may include, for example, at least 3200 polynucleotide probes, or, e.g., at least 10,000 polynucleotide probes, or at least 50,000 probes. The probes may be, for example, 9 to 21 nucleotides in length.